Recent discoveries have raised expectations that stem cells may be a source of replacement cells and tissues that are damaged in the course of disease, infection, or because of congenital abnormalities. Various types of putative stem cells differentiate when they divide, maturing into cells that can carry out the unique functions of particular tissues, such as the heart, the liver, or the brain. A particularly important discovery has been the development of pluripotent stem cells, which are thought to have the potential to differentiate into almost any cell type.
Early work on pluripotent stem cells was done in mice (reviewed in Robertson, Meth. Cell Biol. 75:173, 1997; and Pedersen, Reprod. Fertil. Dev. 6:543, 1994). Mouse stem cells can be isolated from both early embryonic cells and germinal tissue. Desirable characteristics of pluripotent stem cells are that they be capable of indefinite proliferation in vitro in an undifferentiated state, retain a normal karyotype, and retain the potential to differentiate to derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm). Mouse ES cells can be kept in the undifferentiated cells simply by having leukemia inhibitory factor (LIF) in the medium (Williams et al., Nature. 15:684, 1988). Thus, they can be genetically altered and drug selected in culture systems not containing feeder cells.
The development of preparations of human pluripotent stem cells has involved overcoming a number of technical difficulties, which makes them more challenging to grow and manipulate than mouse cells.
Thomson et al. (U.S. Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA 92:7844, 1995) were the first to successfully isolate and propagate pluripotent stem cells from primates. They subsequently derived human embryonic stem (hES) cell lines from human blastocysts (Science 282:114, 1998). Gearhart and coworkers derived human embryonic germ (hEG) cell lines from fetal gonadal tissue (Shambloff et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998; and U.S. Pat. No. 6,090,622). Both hES and hEG cells are capable of long-term proliferation in vitro without differentiating, they have a normal karyotype, and they remain capable of producing a number of different cell types. Because of this, they hold considerable promise for use in human therapy, acting as a reservoir for regeneration of almost any tissue compromised by genetic abnormality, trauma, or a disease condition.
Thomson et al. teach that human ES cells require culturing on feeder cells in order to prevent them from differentiating. This represents a significant challenge to the usual methods of making genetically altered cells.